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The structure and function of oxidized albumin in hemodialysis patients: Its role in elevated oxidative stress via neutrophil burst
BBRC Biochemical and Biophysical Research Communications 334 (2005) 1322–1328 www.elsevier.com/locate/ybbrc
The structure and function of oxidized albumin in hemodialysis patients: Its role in elevated oxidative stress via neutrophil burst Katsumi Mera a,1, Makoto Anraku a,1, Kenichiro Kitamura b, Keisuke Nakajou a, Toru Maruyama a, Masaki Otagiri a,* a
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan b Department of Nephrology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8655, Japan Received 3 July 2005 Available online 18 July 2005
Abstract Oxidized albumin is a reliable marker of oxidative stress in hemodialysis (HD) patients. However, oxidized albumin in vivo and its possible clinical significance has been rarely investigated. In the present study, the qualitative modification of albumin in HD patients (n = 20) was examined and their results were compared with healthy age-matched controls (n = 10). The increase in plasma protein carbonyl levels in HD patients was largely due to an increase in oxidized albumin. Human serum albumin (HSA) of HD patients, HSA of HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. Spectroscopic analysis confirmed that the HD-HSA samples contained higher levels of carbonyls than Normal-HSA. An HPLC analysis also suggested that the state of the purified HSA used throughout the experiments accurately reflects the redox state of albumin in blood. HD-HSA was found to have a decreased the antioxidant activity, and was able to trigger the oxidative burst of human neutrophils, compared to Normal-HSA. HD-HSA was conformationally altered, with its hydrophobic regions more exposed and to have a negative charge. In binding experiments, HD-HSA showed impaired Site II-ligand binding capabilities. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HD patients. 2005 Elsevier Inc. All rights reserved. Keywords: Human serum albumin; Oxidation; Neutrophil burst; Hemodialysis patients; Carbonyl groups; Conformational changes; Hydrophobicity; Net charge; Ligand binding; HPLC analysis
The oxidative modification of proteins and lipids has been implicated in the etiology of numerous disorders and diseases [1,2]. Oxidatively modified plasma proteins can serve as important in vivo biomarkers of oxidative stress. Proteins are better candidates than plasma lipids for use in detecting specific pathways of oxidative stress, due to the accessibility of plasma protein for sampling, their relatively long half-lives, and their well-defined biochemical pathways. Since extracellular fluids contain only small amounts of antioxidant enzymes, it has been proposed that the *
1
Corresponding author. Fax: +81 96 362 7690. E-mail address: [email protected] (M. Otagiri). These two authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.035
major extracellular antioxidants are proteins and circulating albumin is a major antioxidant in plasma [3]. The in vitro oxidation of amino acid residues leads to protein degradation, aggregation, and cross-linking. In contrast, significant evidence for the presence of ROSmediated protein damage in vivo and its possible clinical significance is not currently available. Witko-Sarsat et al. [4] reported the presence of elevated levels of oxidized protein products, termed advanced oxidation protein products (AOPP) such as oxidized albumin, in the plasma of dialysis patients. It has been well documented that human serum albumin (HSA) is quite vulnerable to reactive oxygen species (ROS) [5]. Therefore, HSA is continuously exposed to oxidative stress, so that alterations of the conformation and function of HSA could
K. Mera et al. / Biochemical and Biophysical Research Communications 334 (2005) 1322–1328
occur, resulting in modification of its biological properties. In recent years, activated phagocytes have been reported to be a major source of reactive oxidants and to play a fundamental role in host defense [6,7]. They contain the heme enzyme myeloperoxidase (MPO) which catalyzes the reaction of chloride ion with hydrogen peroxide (H2O2) to generate large amounts of hypochlorous acid (HOCl), a powerful oxidizing and chlorinating agent produced by neutrophils [8–10]. Treatment of HSA with HOCl in vitro and in vivo-generated AOPP have been reported to trigger oxidative bursts in neutrophils as well as in monocytes, thereby appearing to act as true inflammatory mediators [11,12]. The possibility that excessively oxidized albumin in plasma increases the production of ROS by stimulating neutrophils, cannot be excluded. Therefore, very excessively oxidized HSA, could also play an important role as a pro-oxidant in dialysis patients. We previously demonstrated that oxidative stress in HD patients is manifested by an increase in plasma protein oxidation that is characterized by thiol group oxidation and the formation of carbonyl groups on proteins. We also showed that HSA is the major plasma protein target of oxidative stress in uremia, and that increased levels of carbonyl compounds are correlated with the oxidation of albumin in uremic patients [13]. Therefore, serum albumin is an important protein that has direct protective effects. These effects may be also based on a variety of biological mechanisms. In the present study, we further investigated the role of albumin oxidation and neutrophil oxidative burst in HD patients and compared the results with age- and gender-matched control subjects. An evaluation of the effects of oxidative stress on the structural and functional properties of HSA was also attempted.
Materials and methods Patients The protocol used in this study was approved by the institutional review board and informed consent was obtained from all subjects. A total of 30 subjects were enrolled: 20 stable HD patients (10 men, 10 women) aged 36–87 years, with a duration of dialysis ranging from 1 to 9 years, and 10 age- and gender-matched healthy control subjects. End-stage renal failure in HD patients was caused by glomerulonephritis (n = 5), nephrosclerosis (n = 2) or diabetic nephropathy (n = 13). At the time of enrollment, all HD patients had been receiving regular bicarbonate hemodialysis therapy (4–5 h/session, three times per week) using high-flux polysulfone hollow-fiber dialyzers. The profiles of healthy controls and HD patients with or without diabetes are summarized in Table 1. Purified albumin from healthy control and HD patients HSA samples were isolated by polyethylene glycol fractionation of blood plasma followed by chromatography on a blue Sepharose CL-6B column (Amersham-Pharmacia, Uppsala, Sweden) [14]. The
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Table 1 Characteristics of the normal and patient groups
Age (years) Gender (M/F) Diabetes/nondiabetes Creatinine (mg/dL)
Normal subjects (N = 10)
HD patients (N = 20)
67.8 ± 1.8 6/4 — 0.85 ± 0.2
62.8 ± 12.7 10/10 13/7 10.6 ± 2.6*
Values are expressed means ± SD. * P < 0.01.
samples were then dialyzed against deionized water for 48 h at 4 C, followed by lyophilization. The purity of the HSA samples was at least 95%, and the percentage of dimers did not exceed 7%, as evidenced by SDS–PAGE and native-PAGE, respectively. The long-chain fatty acid contents of isolated HSA samples were determined using the copper triethanolamine method [15]. There was no significant change of the long-chain fatty acid contents in purified albumin from healthy controls and HD patients. Determination of serum protein and purified albumin oxidation Chromatographic analysis of albumin in normal subjects and HD patients. HSA is a mixture of mercaptalbumin (HMA; reduced form) and nonmercaptalbumin (HNA; oxidized form). HMA contains one highly reactive sulfhydryl group at position 34 (Cys-34), while other serum proteins contain little or none. HNA is comprised of at least three types of molecules. The major HNA component is a mixed disulfide with cysteine or glutathione (HNA-1). The other is a more highly oxidized product than the mixed disulfide, in which the thiol group has been oxidized to the sulfenic (SOH), sulfinic (SO2H), and sulfonic (SO3H) states (HNA-2), the proportions of which are extremely small in extracellular fluids [16,17]. The high-performance liquid chromatography (HPLC) analysis of albumin developed by Sogami et al. permits the clean separation of HSA into HMA and HNA, and is used for the determination of the redox state for various pathophysiologic conditions. An HPLC analysis of serum albumin was performed as described in a previous report [13]. Serum samples were immediately frozen immediately after they were drawn, and were stored at 80 C until used for HPLC. HPLC was performed using 5 lL aliquots of each serum sample and a Shodex Asahipak ES-502N column (Showa Denko, Tokyo, Japan; column temperature; 35 ± 0.5 C). The HPLC system consisted of an L-6200 intelligent pump equipped with a gradient programmer and an F-1050 fluorescence detector (Jasco, Tokyo, Japan). Elution was performed using a linear gradient with ethanol concentrations increasing from 0% to 5% with the serum dissolved in a mixture of 0.05 mol/L sodium acetate and 0.40 mol/L sodium sulfate (pH 4.85) at a flow rate of 1.0 mL/min. From the HPLC profiles of HSA, the value of each albumin fraction [f(HMA), f(HNA-1), and f(HNA-2)] was estimated by dividing the area of each fraction by the total area corresponding to HSA. Total plasma protein and individual plasma carbonyl contents measurement. Plasma protein carbonyl content was determined using the method of Climent et al. [18]. Biological properties of Normal- and HD-HSA Radical scavenging ability of Normal- and HD-HSA. The radical scavenging activity of Normal- and HD-HSA (10 lM) was determined from the decrease in the absorbance of 1,1 0 -diphenyl-2-picrylhydrazyl (DPPH) radicals at 517 nm due to their scavenging of an unpaired electron of the stable DPPH radical in a mixture of 10 mL ethanol, 10 mL of 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (pH 5.5), and 5 mL of 0.5 mM DPPH in ethanol [19–21].
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Measurement of neutrophil respiratory burst. Neutrophils were isolated from heparinized peripheral blood of healthy donors using Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation. The purity of the PMN preparations routinely exceeded 95%, and cell viability, as determined by propidium iodide staining, was at least 98%. Accumulation of dihydrorhodamine 123 (DRD) in a suspension of neutrophils was measured using a flow cytometer, monitoring the fluorescence at 526 nm [22]. Suspensions of neutrophils (1 · 106 cells) were incubated with 5 lM DRD for 15 min at 37 C in serum-free medium. After DRD incubation, the neutrophil suspension was centrifuged and washed to remove unincorporated probe. The cells were then treated with various concentrations of albumin medium for 1 h at 37 C, and were then placed on ice. The mean fluorescence intensity of rhodamine (RD) in the cells was determined using a flow cytometer (FACS Calibur; Becton–Dickinson Biosciences, Franklin Lakes, NJ). Structural and functional properties of Normal- and HD-HSA CD measurements. These measurements were performed using a Jasco J-720 type spectropolarimeter (Jasco, Tokyo, Japan) at 25 C. Far-UV spectra were recorded at a protein concentration of 20 lM in 67 mM sodium phosphate buffer (pH 7.4) using 1 mm quartz cells. Effective protein hydrophobicity. The effective hydrophobicity of all the albumins (1 lM), dissolved in a 67 mM sodium phosphate buffer (pH 7.4), was probed with 4,4 0 -dianilino-1,1 0 -binaphthyl-5,5 0 -disulfonic acid (bis-ANS) (10 lM) at 25 C. The compound was excited at 394 nm and fluorescence spectra were recorded on a Jasco FP-770 fluorescence spectrometer (Tokyo, Japan) using 1 cm quartz cells. Changes in protein net charge. Changes in the net charge of albumin were evaluated by a modification of the capillary electrophoresis method described by Pande et al. [23]. One milliliter of an HSA sample (2 lM) was run in 100 mM borate buffer (pH 8.5 and 20 C), and the migration time was determined by capillary electrophoresis, model CE990/990-10 from Jasco (Tokyo, Japan). Binding properties. The binding of warfarin (5 lM) and ketoprofen (5 lM) to purified HSA (10 lM) in 67 mM sodium phosphate buffer (pH 7.4 and 25 C) was examined by ultrafiltration. The unbound ligand fractions were separated using an Amicon MPS-1 micropartition system with YMT ultrafiltration membranes by centrifugation (2000g, 40 min). The concentration of unbound ligand was determined by HPLC [14]. The unbound fraction (%) was calculated as follows: Unbound fractionð%Þ ¼ ½ligand concentration in filtered fraction=total ligand concentrationðbefore ultrafiltrationÞ 100.
Statistics The statistical significance of collected data was evaluated using the ANOVA analysis followed by Newman–Keuls method for more than 2
means. A value of P < 0.05 was considered to indicate statistical significance. The results are reported as the mean ± SD.
Results Determination of serum protein and purified albumin oxidation The oxidation of a protein typically results in an increase in carbonyl content. This increase is due to the oxidation of Lys, Arg, or Pro residues. Plasma protein carbonyl contents were significantly increased in HD patients, compared with normal subjects. Data for the carbonyl content were as follows: HD patients groups, 3.12 ± 1.11 nmol/mg protein, n = 20; controls groups, 2.1 ± 0.34 nmol/mg protein, n = 10 (P < 0.01) (Table 2). In a previous study, we determined the redox states of HD patients during oxidative stress, with emphasis on the oxidation of serum albumin [13]. In the present study, using HPLC, we determined the oxidation status of the Cys-34 residues in albumin. Table 2 shows typical HPLC profiles for serum albumin from healthy subjects and HD patients. f(HMA) was substantially decreased and both f(HNA-1) and f(HNA-2) were significantly increased in HD patients compared with healthy subjects (P < 0.01, Table 2). These results suggest that uremia results in an increase in the oxidized serum albumin levels, via oxidative stress. Further, to investigate the extent of alterations in the biological properties of HSA in HD patients, HSA of HD patients (HD-HSA), and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. The albumin carbonyl contents of the purified fraction were significantly increased in HD patients. Data for the carbonyl content of purified HSA were as follows: HD-HSA, 2.86 ± 0.45 nmol/mg protein, n = 20; Normal-HSA, 2.13 ± 0.14 nmol/mg protein, n = 10, P < 0.01 (Table 2). HD-HSA samples also showed a markedly increased HNA ratio, compared with Normal-HSA (P < 0.01, Table 2). In addition, the HNA/HMA ratio of purified HSA was closely correlated with the HNA/HMA of plasma (R = 0.952, P < 0.01;
Table 2 Determination of serum protein and purified albumin oxidation Serum protein
Carbonyl contents (nmol/mg protein) f(HMA) (%) f(HNA-1) (%) f(HNA-2) (%) Values are expressed means ± SD. * P < 0.01 as compared with Normal subjects. ** P < 0.01 as compared with Normal-HSA.
Purified HSA
Normal subjects
HD patients
Normal-HSA
HD-HSA
2.10 ± 0.34 53.6 ± 6.4 38.7 ± 6.3 7.7 ± 0.9
3.12 ± 1.11* 40.4 ± 8.7* 49.7 ± 8.0* 9.9 ± 1.4*
2.13 ± 0.14 46.6 ± 4.9 42.0 ± 4.4 11.4 ± 0.3
2.86 ± 0.45** 32.7 ± 6.8** 54.4 ± 7.1** 12.9 ± 0.7**
K. Mera et al. / Biochemical and Biophysical Research Communications 334 (2005) 1322–1328
Structural and functional properties of Normal- and HD-HSA
Scavenged DPPH (%)
Structural properties of Normal- and HD-HSA The structural properties of Normal- and HD-HSA were examined by a variety of methods. Thus, Fig. 3A 25 20
*
15
*
10 5 0
2.5
5
10
20
MFI (AU)
Number of cells
(2) 80
8.0
7.0 40 0
6.0 100
101
102
Normal-HSA
Relative fluorescence
HD-HSA
Fig. 2. ROS production by neutrophils incubated with purified HSA. (1) Normal-HSA, (2) HD-HSA. *P < 0.05, compared with NormalHSA.
A -1 2
B
10
Fluorescence intensity (AU)
Neutrophil respiratory burst It was recently reported that in vitro-oxidized albumin up-regulates ROS generation in neutrophil suspensions [4]. In order to directly assess whether purified HSA had the ability to induce oxidative stress in a neutrophil suspension, we used the DRD method and a FACS analysis. The levels of ROS were higher for HD-HSA than for Normal-HSA (Fig. 2) (P < 0.05). These results show that HD-HSA was not able to decrease the antioxidant activity, but also to trigger the oxidative burst of human neutrophils.
9.0
120
(deg. cm . dmol )
Radical scavenging ability of Normal- and HD-HSA The effects of Normal- and HD-HSA were examined by monitoring the time-dependent change in the absorbance of DPPH radicals. As shown in Fig. 1, Normal-HSA scavenged DPPH radicals, and its ability to scavenge proceeded more fastly than HD-HSA. HDHSA scavenged about 15% of DPPH radicals even after 20 min. In contrast, the effect of Normal-HSA proceeded more rapidly, about 20% of the DPPH radicals were scavenged within 20 min. This result shows that the radical scavenging activity of Normal-HSA was significantly greater than that of HD-HSA.
(1)
160
-3
Physiological properties of Normal- and HD-HSA
*
200
[ θ] 10
data not shown). These results suggest that the state of purified HSA accurately reflects the redox state of albumin in the blood.
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5 0 -5 -10 -15 -20
(2)
× -25 -30 200
(1) 210
220
230
240
Wavelength (nm)
250
2700
(2) 2500
(1) 2300
2100 460
480
500
520
540
Wavelength (nm)
Fig. 3. (A) Purified HSA far-UV CD spectra of normal subjects and HD patients. (1) Normal-HSA, (2) HD-HSA. The spectra are the average of three determinations. (B) Effect of uremia on the fluorescence of purified HSA-bound bis-ANS. (1) Normal-HSA, (2) HDHSA. The spectra are the average of three determinations.
shows Far-UV CD spectra. As can be seen in Fig. 3A, the characteristics of the CD spectrum of HD-HSA were slightly different from that obtained for Normal-HSA. The effect of oxidation on the exposure of hydrophobic areas was also examined by using the fluorescence probe bis-ANS. The results obtained (Fig. 3B) indicate that the conformation of HD-HSA in particular, involves an increase in accessible hydrophobic regions, compared with Normal-HSA. The net charges of HD- and NormalHSA were investigated by determining their migration times in capillary electrophoresis. The migration time for HD-HSA (25.65 ± 0.52 min) was increased slightly compared with that of Normal-HSA (24.84 ± 0.46 min) (P < 0.01). These results suggest that HD-HSA also underwent conformational changes, hydrophobic regions were more exposed and negative charge on the molecule was increased.
Time (min) Fig. 1. Radical scavenging ability of Normal- and HD-HSA. Normaland HD-HSA at a final concentration of 20 lM were added to a suspension of ethanol, in which DPPH radicals were incorporated (final concentration of 20 lM), in 175 mM KCl and 10 mM Mes buffer (pH 7.4) at 25 C. Changes in the amount of DPPH radicals in the suspension were monitored as a decrease in absorbance at 517 nm. Changes in DPPH radicals scavenged relative to total DPPH radicals with time are shown. *P < 0.05, compared with Normal-HSA.
Binding property of Normal- and HD-HSA The unique ligand binding properties of HSA can, to a great extent, be explained by the presence of two major binding sites, Site I and Site II [24], which are located within specialized cavities in subdomains IIA and IIIA, respectively [25]. The potential effect of oxidation on these sites was examined by using warfarin and ketopro-
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fen as representative ligands. As seen from Table 3, the high-affinity binding of warfarin, which takes place at Site I [26] was little changed, but the high affinity binding of the Site II-ligand ketoprofen [27] was significantly reduced in HD-HSA, compared with Normal-HSA.
Discussion Apart from the low-molecular weight antioxidants, the antioxidant activity in human blood plasma relies mainly on proteins such as albumin [28]. In plasma, the free thiol group is quantitatively the most important scavenger of oxidants. Furthermore, the formation of carbonyl groups in plasma albumin, as previously demonstrated by Himmelfarb and Mcmonagle [28], involves in basic amino acid residues as well as Cys-34 with free thiol group, which is also oxidized. In the previous study, using a Western blot immunoassay, we demonstrated that the carbonylation of albumin accounts for nearly all of the excess plasma protein oxidation in HD patients [13]. We also found that the f(HMA) value in albumin from HD patients was less than 80% of the f(HMA) value in albumin from healthy controls, as indicated by HPLC analysis (Table 2). Since albumin is the most abundant plasma protein, it could play a major role as an antioxidant in plasma at least by thiol oxidation and carbonyl formation. In this context, we expected that the characterization of oxidation status of serum albumin would provide, not only useful information regarding the redox state of the human body, but also alterations in the conformation and function of HSA which may result in modifications of its biological properties. In HD patients with increased oxidative stress, the oxidative modification of plasma proteins especially albumin appear to be more extensive and may be one of the pathological conditions associated with the widespread vascular complications that are frequently observed in HD patients. To investigate the extent of the alterations in the biological properties of HSA in HD patients, HSA from HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified with blue Sepharose CL-6B column chromatography. The retention time and chromatogram of the purified albumin and plasma albumin were found to be highly correlated (Table 2). Thus, the HNA/HMA ratio of purified HSA can be used as an index for the HNA/HMA ratio of plasma (R = 0.952, P < 0.01). The high correlation between the HNA/HMA value of plasma and purified HSA suggest that the state of purified HSA used throughout the experiments accurately reflects the redox state of albumin in the blood. We first focused on the antioxidant activity of albumin because oxidative stress is thought to play a significant role in the pathogenesis of many diseases,
including atherosclerosis [29,30]. There is now ample evidence to suggest that albumin, as the main circulating protein, is a quantitatively important antioxidant in the blood and extracellular fluids [31,32]. Our present studies are in full agreement with this view, Normal-HSA offered significant protection against free radicals, compared to HD-HSA (Fig. 1). These antioxidant effects were found to be concentration-dependent (data not shown), consistent with a beneficial effect of high albumin levels in humans. This in turn suggests that excessively oxidized albumin, which acts as a pro-oxidant, may increase cardiovascular complications in HD patients. In these patients recurrent blood interactions with bioincompatible dialysis membranes trigger neutrophil activation and the subsequent generation of highly reactive oxygen species (ROS) including O2 and its derivatives (H2O2, OH, and 1O2) via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and OCl via a MPO-dependent reaction between chloride and H2O2 [4]. To test the hypothesis that the increased oxidative stress in blood of HD patients is caused by oxidized albumin, we examined the effects of incubating neutrophils with purified albumin from Normal- or HD-HSA. Purified albumin from HD patients (HD-HSA) induced oxidative stress via a neutrophil respiratory burst (Fig. 2). In previous studies, excess AOPP-modified albumin, was found to be associated with a high level of respiratory burst [12], but the samples used in those studies were produced by chemical modification in vitro. In the present studies, HD-HSA, isolated from uremia patients was found to have been modified in vivo. Treatment of neutrophils with purified albumin from HD patients induced oxidative stress. Therefore, HD-HSA was, found not only to have a decreased antioxidant activity, but also able to trigger the oxidative burst of human neutrophils, compared with Normal-HSA. These results suggest that HSA is continuously exposed to oxidative stress, so much so that alterations in its biological properties that could result in the conformational, functional changes of HSA occur. Therefore, the significance of conformational changes of purified HSA on its functional properties were examined. Slight decreases in a-helical content accompanied by tertiary conformational changes were observed for HD-HSA (data not shown). These changes, which could
Table 3 Binding of warfarin and ketoprofen to Normal-HSA and HD-HSA Free fraction (%)
Normal-HSA HD-HSA
Warfarin
Ketoprofen
19.6 ± 1.9 23.3 ± 2.0
5.97 ± 0.75 9.37 ± 1.36*
Values are expressed means ± SD. * P < 0.05.
K. Mera et al. / Biochemical and Biophysical Research Communications 334 (2005) 1322–1328
be observed by near-UV CD resulted in an increased exposure of hydrophobic regions of the protein (Fig. 3). Changes in net charge on the HSA surface were also observed. These results suggest that the increase of negative charge reflects oxidation of basic amino acid residues in albumin. In plasma, all amino acids in the protein are susceptible to oxidative modification by oxidants such as hydroxyl radicals and hypochlorous acid. Among them, amino acids such as cysteine, histidine, lysine, and arginine are more vulnerable to oxidation [33]. Modification of these residues results in conformational changes in cases of uremia. The high-affinity binding of warfarin and ketoprofen was studied in order to determine whether the drug binding properties of HSA had been affected by uremia. In this experiment, the drug binding properties of HDHSA were found to be reduced. The decreased ketoprofen binding to HD-HSA is most probably caused by conformational changes involving Site II in subdomain IIIA [24]. The decreased binding to HD-HSA might be also caused by the oxidation of 410Arg. Ahmed et al. [34] recently suggested that 410Arg is a target site for oxidative stress. Using an anti-carboxyl methyl-Lysine and an anti-hydro-imidazolone antibody to identify advanced glycation endproducts, we also found that HD-HSA could be recognized by this antibody. This indicates that Lys and Arg residues in HD-HSA were modified (data not shown), causing structural changes due to oxidative stress in HD patients. In addition, this alteration may further aggravate the pre-existing elevated concentration of unbound free drugs in HD patients. Therefore, the structural alteration of HSA in uremia results in the loss of some important properties of HSA. In conclusion, the present study has shown that the oxidative modification of HSA in HD patients may lead to alterations in the conformation as well as the biological properties of HSA. The present study further propose that, in addition to its serum concentration, the quality of HSA molecules may be not only a crucial factor affecting its protective effects but also a risk factor as a pro-oxidant in HD patients. The present study provides evidence to suggest that isolated HSA in hemodialyzed patients has, not only reduced structural and functional properties, but also acts as a mediator of the neutrophil activation state associated with chronic uremia.
Acknowledgments Authors thank Dr. Shunichi Sakaguchi (Midorigaoka Clinic, Kumamoto, Japan) for blood sample collection from HD patients.This work was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (15790432 to K.K., 14370759 and
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14657618 to M.O.), the Salt Science Research Foundation (0330 to K.K.), and Japan Heart Foundation Research Grant (to K.K.)
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studies on the resolution of human mercapt- and nonmercaptalbumin and on human serum albumin in the elderly by highperformance liquid chromatography, Int. J. Pept. Protein Res. 3 (1985) 435–442. I. Climent, L. Tsai, R.L. Levine, Derivatization of gammaglutamyl semialdehyde residues in oxidized proteins by fluoresceinamine, Anal. Biochem. 182 (1989) 226–232. H. Sassa, Y. Takaishi, H. Terada, The triterpene celastrol as a very potent inhibitor of lipid peroxidation in mitochondria, Biochem. Biophys. Res. Commun. 172 (1990) 890–897. H. Sassa, K. Kogure, Y. Takaishi, H. Terada, Structural basis of potent antiperoxidation activity of the triterpene celastrol in mitochondria: effect of negative membrane surface charge on lipid peroxidation, Free Radic. Biol. Med. 17 (1994) 201–207. K. Kogure, S. Goto, K. Abe, C. Ohiwa, M. Akasu, H. Terada, Potent antiperoxidation activity of the bisbenzylisoquinoline alkaloid cepharanthine: the amine moiety is responsible for its pH-dependent radical scavenge activity, Biochim. Biophys. Acta 1426 (1999) 133–142. H. Nakajima, M. Takenaka, J.Y. Kaimori, T. Hamano, H. Iwatani, T. Sugaya, T. Ito, M. Hori, E. Imai, Activation of the signal transducer and activator of transcription signaling pathway in renal proximal tubular cells by albumin, J. Am. Soc. Nephrol. 15 (2004) 276–285. P.G. Pande, R.V. Nellore, H.R. Bhagat, Optimization and validation of analytical conditions for bovine serum albumin using capillary electrophoresis, Anal. Biochem. 204 (1992) 103–106. G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824–832.
[25] D.C. Carter, J.X. Ho, Structure of serum albumin, Adv. Protein Chem. 45 (1994) 153–203. [26] T. Peters Jr, All about Albumin, Biochemistry, Genetics, and Medical Applications, Academic Press, San Diego, 1996. [27] V.T.G. Chuang, A. Kuniyasu, H. Nakayama, Y. Matsushita, S. Hirono, M. Otagiri, Helix 6 of subdomain IIIA of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety, Biochim. Biophys. Acta 1434 (1999) 18–30. [28] J. Himmelfarb, E. Mcmonagle, Albumin is the major plasma protein target of oxidant stress in uremia, Kidney Int. 60 (2001) 358–363. [29] J.P. Kehrer, Free radicals as mediators of tissue injury and disease, Crit. Rev. Toxicol. 23 (1993) 21–48. [30] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. USA 90 (1993) 7915–7922. [31] D.D. Wayner, G. Burton, K.U. Ingold, S.J. Locke, Quantitative measurement of total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation, FEBS Lett. 187 (1985) 33–37. [32] B. Halliwell, J.M. Gutteridge, The antioxidants of human extracellular fluids, Arch. Biochem. Biophys. 280 (1990) 1–8. [33] R.L. Levine, J.A. Williams, E.R. Stadtman, E. Shacter, Carbonyl assays for determination of oxidatively modified proteins, Methods Enzymol. 233 (1994) 346–363. [34] N. Ahmed, D. Dobler, M. Dean, P.J. Thornalley, Peptide mapping identifies hotspot site of modification in human serum albumin by methylglyoxal involved in ligand binding and esterase activity, J. Biol. Chem. 280 (2005) 5724–5732.
The structure and function of oxidized albumin in hemodialysis patients: Its role in elevated oxidative stress via neutrophil burst Katsumi Mera a,1, Makoto Anraku a,1, Kenichiro Kitamura b, Keisuke Nakajou a, Toru Maruyama a, Masaki Otagiri a,* a
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan b Department of Nephrology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8655, Japan Received 3 July 2005 Available online 18 July 2005
Abstract Oxidized albumin is a reliable marker of oxidative stress in hemodialysis (HD) patients. However, oxidized albumin in vivo and its possible clinical significance has been rarely investigated. In the present study, the qualitative modification of albumin in HD patients (n = 20) was examined and their results were compared with healthy age-matched controls (n = 10). The increase in plasma protein carbonyl levels in HD patients was largely due to an increase in oxidized albumin. Human serum albumin (HSA) of HD patients, HSA of HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. Spectroscopic analysis confirmed that the HD-HSA samples contained higher levels of carbonyls than Normal-HSA. An HPLC analysis also suggested that the state of the purified HSA used throughout the experiments accurately reflects the redox state of albumin in blood. HD-HSA was found to have a decreased the antioxidant activity, and was able to trigger the oxidative burst of human neutrophils, compared to Normal-HSA. HD-HSA was conformationally altered, with its hydrophobic regions more exposed and to have a negative charge. In binding experiments, HD-HSA showed impaired Site II-ligand binding capabilities. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HD patients. 2005 Elsevier Inc. All rights reserved. Keywords: Human serum albumin; Oxidation; Neutrophil burst; Hemodialysis patients; Carbonyl groups; Conformational changes; Hydrophobicity; Net charge; Ligand binding; HPLC analysis
The oxidative modification of proteins and lipids has been implicated in the etiology of numerous disorders and diseases [1,2]. Oxidatively modified plasma proteins can serve as important in vivo biomarkers of oxidative stress. Proteins are better candidates than plasma lipids for use in detecting specific pathways of oxidative stress, due to the accessibility of plasma protein for sampling, their relatively long half-lives, and their well-defined biochemical pathways. Since extracellular fluids contain only small amounts of antioxidant enzymes, it has been proposed that the *
1
Corresponding author. Fax: +81 96 362 7690. E-mail address: [email protected] (M. Otagiri). These two authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.035
major extracellular antioxidants are proteins and circulating albumin is a major antioxidant in plasma [3]. The in vitro oxidation of amino acid residues leads to protein degradation, aggregation, and cross-linking. In contrast, significant evidence for the presence of ROSmediated protein damage in vivo and its possible clinical significance is not currently available. Witko-Sarsat et al. [4] reported the presence of elevated levels of oxidized protein products, termed advanced oxidation protein products (AOPP) such as oxidized albumin, in the plasma of dialysis patients. It has been well documented that human serum albumin (HSA) is quite vulnerable to reactive oxygen species (ROS) [5]. Therefore, HSA is continuously exposed to oxidative stress, so that alterations of the conformation and function of HSA could
K. Mera et al. / Biochemical and Biophysical Research Communications 334 (2005) 1322–1328
occur, resulting in modification of its biological properties. In recent years, activated phagocytes have been reported to be a major source of reactive oxidants and to play a fundamental role in host defense [6,7]. They contain the heme enzyme myeloperoxidase (MPO) which catalyzes the reaction of chloride ion with hydrogen peroxide (H2O2) to generate large amounts of hypochlorous acid (HOCl), a powerful oxidizing and chlorinating agent produced by neutrophils [8–10]. Treatment of HSA with HOCl in vitro and in vivo-generated AOPP have been reported to trigger oxidative bursts in neutrophils as well as in monocytes, thereby appearing to act as true inflammatory mediators [11,12]. The possibility that excessively oxidized albumin in plasma increases the production of ROS by stimulating neutrophils, cannot be excluded. Therefore, very excessively oxidized HSA, could also play an important role as a pro-oxidant in dialysis patients. We previously demonstrated that oxidative stress in HD patients is manifested by an increase in plasma protein oxidation that is characterized by thiol group oxidation and the formation of carbonyl groups on proteins. We also showed that HSA is the major plasma protein target of oxidative stress in uremia, and that increased levels of carbonyl compounds are correlated with the oxidation of albumin in uremic patients [13]. Therefore, serum albumin is an important protein that has direct protective effects. These effects may be also based on a variety of biological mechanisms. In the present study, we further investigated the role of albumin oxidation and neutrophil oxidative burst in HD patients and compared the results with age- and gender-matched control subjects. An evaluation of the effects of oxidative stress on the structural and functional properties of HSA was also attempted.
Materials and methods Patients The protocol used in this study was approved by the institutional review board and informed consent was obtained from all subjects. A total of 30 subjects were enrolled: 20 stable HD patients (10 men, 10 women) aged 36–87 years, with a duration of dialysis ranging from 1 to 9 years, and 10 age- and gender-matched healthy control subjects. End-stage renal failure in HD patients was caused by glomerulonephritis (n = 5), nephrosclerosis (n = 2) or diabetic nephropathy (n = 13). At the time of enrollment, all HD patients had been receiving regular bicarbonate hemodialysis therapy (4–5 h/session, three times per week) using high-flux polysulfone hollow-fiber dialyzers. The profiles of healthy controls and HD patients with or without diabetes are summarized in Table 1. Purified albumin from healthy control and HD patients HSA samples were isolated by polyethylene glycol fractionation of blood plasma followed by chromatography on a blue Sepharose CL-6B column (Amersham-Pharmacia, Uppsala, Sweden) [14]. The
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Table 1 Characteristics of the normal and patient groups
Age (years) Gender (M/F) Diabetes/nondiabetes Creatinine (mg/dL)
Normal subjects (N = 10)
HD patients (N = 20)
67.8 ± 1.8 6/4 — 0.85 ± 0.2
62.8 ± 12.7 10/10 13/7 10.6 ± 2.6*
Values are expressed means ± SD. * P < 0.01.
samples were then dialyzed against deionized water for 48 h at 4 C, followed by lyophilization. The purity of the HSA samples was at least 95%, and the percentage of dimers did not exceed 7%, as evidenced by SDS–PAGE and native-PAGE, respectively. The long-chain fatty acid contents of isolated HSA samples were determined using the copper triethanolamine method [15]. There was no significant change of the long-chain fatty acid contents in purified albumin from healthy controls and HD patients. Determination of serum protein and purified albumin oxidation Chromatographic analysis of albumin in normal subjects and HD patients. HSA is a mixture of mercaptalbumin (HMA; reduced form) and nonmercaptalbumin (HNA; oxidized form). HMA contains one highly reactive sulfhydryl group at position 34 (Cys-34), while other serum proteins contain little or none. HNA is comprised of at least three types of molecules. The major HNA component is a mixed disulfide with cysteine or glutathione (HNA-1). The other is a more highly oxidized product than the mixed disulfide, in which the thiol group has been oxidized to the sulfenic (SOH), sulfinic (SO2H), and sulfonic (SO3H) states (HNA-2), the proportions of which are extremely small in extracellular fluids [16,17]. The high-performance liquid chromatography (HPLC) analysis of albumin developed by Sogami et al. permits the clean separation of HSA into HMA and HNA, and is used for the determination of the redox state for various pathophysiologic conditions. An HPLC analysis of serum albumin was performed as described in a previous report [13]. Serum samples were immediately frozen immediately after they were drawn, and were stored at 80 C until used for HPLC. HPLC was performed using 5 lL aliquots of each serum sample and a Shodex Asahipak ES-502N column (Showa Denko, Tokyo, Japan; column temperature; 35 ± 0.5 C). The HPLC system consisted of an L-6200 intelligent pump equipped with a gradient programmer and an F-1050 fluorescence detector (Jasco, Tokyo, Japan). Elution was performed using a linear gradient with ethanol concentrations increasing from 0% to 5% with the serum dissolved in a mixture of 0.05 mol/L sodium acetate and 0.40 mol/L sodium sulfate (pH 4.85) at a flow rate of 1.0 mL/min. From the HPLC profiles of HSA, the value of each albumin fraction [f(HMA), f(HNA-1), and f(HNA-2)] was estimated by dividing the area of each fraction by the total area corresponding to HSA. Total plasma protein and individual plasma carbonyl contents measurement. Plasma protein carbonyl content was determined using the method of Climent et al. [18]. Biological properties of Normal- and HD-HSA Radical scavenging ability of Normal- and HD-HSA. The radical scavenging activity of Normal- and HD-HSA (10 lM) was determined from the decrease in the absorbance of 1,1 0 -diphenyl-2-picrylhydrazyl (DPPH) radicals at 517 nm due to their scavenging of an unpaired electron of the stable DPPH radical in a mixture of 10 mL ethanol, 10 mL of 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (pH 5.5), and 5 mL of 0.5 mM DPPH in ethanol [19–21].
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Measurement of neutrophil respiratory burst. Neutrophils were isolated from heparinized peripheral blood of healthy donors using Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation. The purity of the PMN preparations routinely exceeded 95%, and cell viability, as determined by propidium iodide staining, was at least 98%. Accumulation of dihydrorhodamine 123 (DRD) in a suspension of neutrophils was measured using a flow cytometer, monitoring the fluorescence at 526 nm [22]. Suspensions of neutrophils (1 · 106 cells) were incubated with 5 lM DRD for 15 min at 37 C in serum-free medium. After DRD incubation, the neutrophil suspension was centrifuged and washed to remove unincorporated probe. The cells were then treated with various concentrations of albumin medium for 1 h at 37 C, and were then placed on ice. The mean fluorescence intensity of rhodamine (RD) in the cells was determined using a flow cytometer (FACS Calibur; Becton–Dickinson Biosciences, Franklin Lakes, NJ). Structural and functional properties of Normal- and HD-HSA CD measurements. These measurements were performed using a Jasco J-720 type spectropolarimeter (Jasco, Tokyo, Japan) at 25 C. Far-UV spectra were recorded at a protein concentration of 20 lM in 67 mM sodium phosphate buffer (pH 7.4) using 1 mm quartz cells. Effective protein hydrophobicity. The effective hydrophobicity of all the albumins (1 lM), dissolved in a 67 mM sodium phosphate buffer (pH 7.4), was probed with 4,4 0 -dianilino-1,1 0 -binaphthyl-5,5 0 -disulfonic acid (bis-ANS) (10 lM) at 25 C. The compound was excited at 394 nm and fluorescence spectra were recorded on a Jasco FP-770 fluorescence spectrometer (Tokyo, Japan) using 1 cm quartz cells. Changes in protein net charge. Changes in the net charge of albumin were evaluated by a modification of the capillary electrophoresis method described by Pande et al. [23]. One milliliter of an HSA sample (2 lM) was run in 100 mM borate buffer (pH 8.5 and 20 C), and the migration time was determined by capillary electrophoresis, model CE990/990-10 from Jasco (Tokyo, Japan). Binding properties. The binding of warfarin (5 lM) and ketoprofen (5 lM) to purified HSA (10 lM) in 67 mM sodium phosphate buffer (pH 7.4 and 25 C) was examined by ultrafiltration. The unbound ligand fractions were separated using an Amicon MPS-1 micropartition system with YMT ultrafiltration membranes by centrifugation (2000g, 40 min). The concentration of unbound ligand was determined by HPLC [14]. The unbound fraction (%) was calculated as follows: Unbound fractionð%Þ ¼ ½ligand concentration in filtered fraction=total ligand concentrationðbefore ultrafiltrationÞ 100.
Statistics The statistical significance of collected data was evaluated using the ANOVA analysis followed by Newman–Keuls method for more than 2
means. A value of P < 0.05 was considered to indicate statistical significance. The results are reported as the mean ± SD.
Results Determination of serum protein and purified albumin oxidation The oxidation of a protein typically results in an increase in carbonyl content. This increase is due to the oxidation of Lys, Arg, or Pro residues. Plasma protein carbonyl contents were significantly increased in HD patients, compared with normal subjects. Data for the carbonyl content were as follows: HD patients groups, 3.12 ± 1.11 nmol/mg protein, n = 20; controls groups, 2.1 ± 0.34 nmol/mg protein, n = 10 (P < 0.01) (Table 2). In a previous study, we determined the redox states of HD patients during oxidative stress, with emphasis on the oxidation of serum albumin [13]. In the present study, using HPLC, we determined the oxidation status of the Cys-34 residues in albumin. Table 2 shows typical HPLC profiles for serum albumin from healthy subjects and HD patients. f(HMA) was substantially decreased and both f(HNA-1) and f(HNA-2) were significantly increased in HD patients compared with healthy subjects (P < 0.01, Table 2). These results suggest that uremia results in an increase in the oxidized serum albumin levels, via oxidative stress. Further, to investigate the extent of alterations in the biological properties of HSA in HD patients, HSA of HD patients (HD-HSA), and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. The albumin carbonyl contents of the purified fraction were significantly increased in HD patients. Data for the carbonyl content of purified HSA were as follows: HD-HSA, 2.86 ± 0.45 nmol/mg protein, n = 20; Normal-HSA, 2.13 ± 0.14 nmol/mg protein, n = 10, P < 0.01 (Table 2). HD-HSA samples also showed a markedly increased HNA ratio, compared with Normal-HSA (P < 0.01, Table 2). In addition, the HNA/HMA ratio of purified HSA was closely correlated with the HNA/HMA of plasma (R = 0.952, P < 0.01;
Table 2 Determination of serum protein and purified albumin oxidation Serum protein
Carbonyl contents (nmol/mg protein) f(HMA) (%) f(HNA-1) (%) f(HNA-2) (%) Values are expressed means ± SD. * P < 0.01 as compared with Normal subjects. ** P < 0.01 as compared with Normal-HSA.
Purified HSA
Normal subjects
HD patients
Normal-HSA
HD-HSA
2.10 ± 0.34 53.6 ± 6.4 38.7 ± 6.3 7.7 ± 0.9
3.12 ± 1.11* 40.4 ± 8.7* 49.7 ± 8.0* 9.9 ± 1.4*
2.13 ± 0.14 46.6 ± 4.9 42.0 ± 4.4 11.4 ± 0.3
2.86 ± 0.45** 32.7 ± 6.8** 54.4 ± 7.1** 12.9 ± 0.7**
K. Mera et al. / Biochemical and Biophysical Research Communications 334 (2005) 1322–1328
Structural and functional properties of Normal- and HD-HSA
Scavenged DPPH (%)
Structural properties of Normal- and HD-HSA The structural properties of Normal- and HD-HSA were examined by a variety of methods. Thus, Fig. 3A 25 20
*
15
*
10 5 0
2.5
5
10
20
MFI (AU)
Number of cells
(2) 80
8.0
7.0 40 0
6.0 100
101
102
Normal-HSA
Relative fluorescence
HD-HSA
Fig. 2. ROS production by neutrophils incubated with purified HSA. (1) Normal-HSA, (2) HD-HSA. *P < 0.05, compared with NormalHSA.
A -1 2
B
10
Fluorescence intensity (AU)
Neutrophil respiratory burst It was recently reported that in vitro-oxidized albumin up-regulates ROS generation in neutrophil suspensions [4]. In order to directly assess whether purified HSA had the ability to induce oxidative stress in a neutrophil suspension, we used the DRD method and a FACS analysis. The levels of ROS were higher for HD-HSA than for Normal-HSA (Fig. 2) (P < 0.05). These results show that HD-HSA was not able to decrease the antioxidant activity, but also to trigger the oxidative burst of human neutrophils.
9.0
120
(deg. cm . dmol )
Radical scavenging ability of Normal- and HD-HSA The effects of Normal- and HD-HSA were examined by monitoring the time-dependent change in the absorbance of DPPH radicals. As shown in Fig. 1, Normal-HSA scavenged DPPH radicals, and its ability to scavenge proceeded more fastly than HD-HSA. HDHSA scavenged about 15% of DPPH radicals even after 20 min. In contrast, the effect of Normal-HSA proceeded more rapidly, about 20% of the DPPH radicals were scavenged within 20 min. This result shows that the radical scavenging activity of Normal-HSA was significantly greater than that of HD-HSA.
(1)
160
-3
Physiological properties of Normal- and HD-HSA
*
200
[ θ] 10
data not shown). These results suggest that the state of purified HSA accurately reflects the redox state of albumin in the blood.
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5 0 -5 -10 -15 -20
(2)
× -25 -30 200
(1) 210
220
230
240
Wavelength (nm)
250
2700
(2) 2500
(1) 2300
2100 460
480
500
520
540
Wavelength (nm)
Fig. 3. (A) Purified HSA far-UV CD spectra of normal subjects and HD patients. (1) Normal-HSA, (2) HD-HSA. The spectra are the average of three determinations. (B) Effect of uremia on the fluorescence of purified HSA-bound bis-ANS. (1) Normal-HSA, (2) HDHSA. The spectra are the average of three determinations.
shows Far-UV CD spectra. As can be seen in Fig. 3A, the characteristics of the CD spectrum of HD-HSA were slightly different from that obtained for Normal-HSA. The effect of oxidation on the exposure of hydrophobic areas was also examined by using the fluorescence probe bis-ANS. The results obtained (Fig. 3B) indicate that the conformation of HD-HSA in particular, involves an increase in accessible hydrophobic regions, compared with Normal-HSA. The net charges of HD- and NormalHSA were investigated by determining their migration times in capillary electrophoresis. The migration time for HD-HSA (25.65 ± 0.52 min) was increased slightly compared with that of Normal-HSA (24.84 ± 0.46 min) (P < 0.01). These results suggest that HD-HSA also underwent conformational changes, hydrophobic regions were more exposed and negative charge on the molecule was increased.
Time (min) Fig. 1. Radical scavenging ability of Normal- and HD-HSA. Normaland HD-HSA at a final concentration of 20 lM were added to a suspension of ethanol, in which DPPH radicals were incorporated (final concentration of 20 lM), in 175 mM KCl and 10 mM Mes buffer (pH 7.4) at 25 C. Changes in the amount of DPPH radicals in the suspension were monitored as a decrease in absorbance at 517 nm. Changes in DPPH radicals scavenged relative to total DPPH radicals with time are shown. *P < 0.05, compared with Normal-HSA.
Binding property of Normal- and HD-HSA The unique ligand binding properties of HSA can, to a great extent, be explained by the presence of two major binding sites, Site I and Site II [24], which are located within specialized cavities in subdomains IIA and IIIA, respectively [25]. The potential effect of oxidation on these sites was examined by using warfarin and ketopro-
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fen as representative ligands. As seen from Table 3, the high-affinity binding of warfarin, which takes place at Site I [26] was little changed, but the high affinity binding of the Site II-ligand ketoprofen [27] was significantly reduced in HD-HSA, compared with Normal-HSA.
Discussion Apart from the low-molecular weight antioxidants, the antioxidant activity in human blood plasma relies mainly on proteins such as albumin [28]. In plasma, the free thiol group is quantitatively the most important scavenger of oxidants. Furthermore, the formation of carbonyl groups in plasma albumin, as previously demonstrated by Himmelfarb and Mcmonagle [28], involves in basic amino acid residues as well as Cys-34 with free thiol group, which is also oxidized. In the previous study, using a Western blot immunoassay, we demonstrated that the carbonylation of albumin accounts for nearly all of the excess plasma protein oxidation in HD patients [13]. We also found that the f(HMA) value in albumin from HD patients was less than 80% of the f(HMA) value in albumin from healthy controls, as indicated by HPLC analysis (Table 2). Since albumin is the most abundant plasma protein, it could play a major role as an antioxidant in plasma at least by thiol oxidation and carbonyl formation. In this context, we expected that the characterization of oxidation status of serum albumin would provide, not only useful information regarding the redox state of the human body, but also alterations in the conformation and function of HSA which may result in modifications of its biological properties. In HD patients with increased oxidative stress, the oxidative modification of plasma proteins especially albumin appear to be more extensive and may be one of the pathological conditions associated with the widespread vascular complications that are frequently observed in HD patients. To investigate the extent of the alterations in the biological properties of HSA in HD patients, HSA from HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified with blue Sepharose CL-6B column chromatography. The retention time and chromatogram of the purified albumin and plasma albumin were found to be highly correlated (Table 2). Thus, the HNA/HMA ratio of purified HSA can be used as an index for the HNA/HMA ratio of plasma (R = 0.952, P < 0.01). The high correlation between the HNA/HMA value of plasma and purified HSA suggest that the state of purified HSA used throughout the experiments accurately reflects the redox state of albumin in the blood. We first focused on the antioxidant activity of albumin because oxidative stress is thought to play a significant role in the pathogenesis of many diseases,
including atherosclerosis [29,30]. There is now ample evidence to suggest that albumin, as the main circulating protein, is a quantitatively important antioxidant in the blood and extracellular fluids [31,32]. Our present studies are in full agreement with this view, Normal-HSA offered significant protection against free radicals, compared to HD-HSA (Fig. 1). These antioxidant effects were found to be concentration-dependent (data not shown), consistent with a beneficial effect of high albumin levels in humans. This in turn suggests that excessively oxidized albumin, which acts as a pro-oxidant, may increase cardiovascular complications in HD patients. In these patients recurrent blood interactions with bioincompatible dialysis membranes trigger neutrophil activation and the subsequent generation of highly reactive oxygen species (ROS) including O2 and its derivatives (H2O2, OH, and 1O2) via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and OCl via a MPO-dependent reaction between chloride and H2O2 [4]. To test the hypothesis that the increased oxidative stress in blood of HD patients is caused by oxidized albumin, we examined the effects of incubating neutrophils with purified albumin from Normal- or HD-HSA. Purified albumin from HD patients (HD-HSA) induced oxidative stress via a neutrophil respiratory burst (Fig. 2). In previous studies, excess AOPP-modified albumin, was found to be associated with a high level of respiratory burst [12], but the samples used in those studies were produced by chemical modification in vitro. In the present studies, HD-HSA, isolated from uremia patients was found to have been modified in vivo. Treatment of neutrophils with purified albumin from HD patients induced oxidative stress. Therefore, HD-HSA was, found not only to have a decreased antioxidant activity, but also able to trigger the oxidative burst of human neutrophils, compared with Normal-HSA. These results suggest that HSA is continuously exposed to oxidative stress, so much so that alterations in its biological properties that could result in the conformational, functional changes of HSA occur. Therefore, the significance of conformational changes of purified HSA on its functional properties were examined. Slight decreases in a-helical content accompanied by tertiary conformational changes were observed for HD-HSA (data not shown). These changes, which could
Table 3 Binding of warfarin and ketoprofen to Normal-HSA and HD-HSA Free fraction (%)
Normal-HSA HD-HSA
Warfarin
Ketoprofen
19.6 ± 1.9 23.3 ± 2.0
5.97 ± 0.75 9.37 ± 1.36*
Values are expressed means ± SD. * P < 0.05.
K. Mera et al. / Biochemical and Biophysical Research Communications 334 (2005) 1322–1328
be observed by near-UV CD resulted in an increased exposure of hydrophobic regions of the protein (Fig. 3). Changes in net charge on the HSA surface were also observed. These results suggest that the increase of negative charge reflects oxidation of basic amino acid residues in albumin. In plasma, all amino acids in the protein are susceptible to oxidative modification by oxidants such as hydroxyl radicals and hypochlorous acid. Among them, amino acids such as cysteine, histidine, lysine, and arginine are more vulnerable to oxidation [33]. Modification of these residues results in conformational changes in cases of uremia. The high-affinity binding of warfarin and ketoprofen was studied in order to determine whether the drug binding properties of HSA had been affected by uremia. In this experiment, the drug binding properties of HDHSA were found to be reduced. The decreased ketoprofen binding to HD-HSA is most probably caused by conformational changes involving Site II in subdomain IIIA [24]. The decreased binding to HD-HSA might be also caused by the oxidation of 410Arg. Ahmed et al. [34] recently suggested that 410Arg is a target site for oxidative stress. Using an anti-carboxyl methyl-Lysine and an anti-hydro-imidazolone antibody to identify advanced glycation endproducts, we also found that HD-HSA could be recognized by this antibody. This indicates that Lys and Arg residues in HD-HSA were modified (data not shown), causing structural changes due to oxidative stress in HD patients. In addition, this alteration may further aggravate the pre-existing elevated concentration of unbound free drugs in HD patients. Therefore, the structural alteration of HSA in uremia results in the loss of some important properties of HSA. In conclusion, the present study has shown that the oxidative modification of HSA in HD patients may lead to alterations in the conformation as well as the biological properties of HSA. The present study further propose that, in addition to its serum concentration, the quality of HSA molecules may be not only a crucial factor affecting its protective effects but also a risk factor as a pro-oxidant in HD patients. The present study provides evidence to suggest that isolated HSA in hemodialyzed patients has, not only reduced structural and functional properties, but also acts as a mediator of the neutrophil activation state associated with chronic uremia.
Acknowledgments Authors thank Dr. Shunichi Sakaguchi (Midorigaoka Clinic, Kumamoto, Japan) for blood sample collection from HD patients.This work was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (15790432 to K.K., 14370759 and
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14657618 to M.O.), the Salt Science Research Foundation (0330 to K.K.), and Japan Heart Foundation Research Grant (to K.K.)
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